Binding protein

ABSTRACT

A functionally unglycosylated transmembrane protein having a molecular weight of about 24 kd which is ubiquitous in human cells and exhibits the same species specificity as hepatitis C virus. The protein is capable of binding to HCV surface proteins and is a putative HCV cellular receptor. As such it has many applications in the fields of diagnosis and treatment of HCV infection and in the design of HCV therapeutics.

FIELD OF THE INVENTION

[0001] The present invention relates to proteins capable of binding theE2 envelope protein of hepatitis C virus (HCV) and to processes forproduction and purification.

[0002] The invention also relates to the use of the proteins in therapyand diagnosis and to pharmaceutical compositions and diagnostic kits forsuch uses. The invention also relates to a process for screeningputative molecules for competition with HCV for receptor binding. Theinvention also relates to an animal model for HCV infection.

BRIEF DESCRIPTION OF THE PRIOR ART

[0003] HCV (previously known as Non-A Non-B hepatitis—NANBV) is apositive sense RNA virus of about 10000 nucleotides with a single openreading frame encoding a polyprotein of about 3000 amino acids. Althoughthe structure of the virus has been elucidated by recombinant DNAtechniques (1, 2), the virus itself has not been isolated and thefunctions of the various viral proteins produced by proteolysis of thepolyprotein have only been inferred by analogy with other similarviruses of similar genomic organisation (3).

[0004] The viral proteins are all available in recombinant form,expressed in a variety of cells and cell types, including yeast,bacteria, insect and mammalian cells (4,5).

[0005] Two proteins, named E1 and E2 (corresponding to amino acids192-383 and 384-750 respectively) have been suggested to be externalproteins of the viral envelope which are responsible for the binding ofvirus to target cells (3).

[0006] HCV research is hindered very considerably by the limited hostrange of the virus. The only reliable animal model for HCV infection isthe chimpanzee and it is not possible to propagate HCV in tissueculture.

[0007] In our copending International patent application PCT/IB95/00692,we describe a method employing flow cytometry to identify cells carryingthe HCV receptor. We have shown that, by labelling cells withrecombinant E2 envelope protein, it is possible to sort cells using flowcytometry, isolating those cells capable of specific binding to the E2and therefore potentially carrying the HCV receptor. Employing thistechnique, we have identified a protein capable of binding to the E2envelope protein of HCV which we believe to be the receptor for HCV,thereby enabling overcoming many problems in the art.

SUMMARY OF THE INVENTION

[0008] According to the present invention, there is provided a proteinhaving a molecular weight of about 24 kD and capable of specificallybinding to a protein of hepatitis C virus, or a functionally equivalentvariant or fragment thereof.

[0009] It will be understood by the skilled person that molecularweights measured as described below using electrophoresis are inherentlysubject to interpretation since they are measured relative to standardmolecular weight markers. However, in the context of this specificationthe expression “24 kd” is unambiguous when read in context, since onlyone such protein is obtained by following the processes described belowwith the defined characteristic of binding to hepatitis C virus.

[0010] A significant characterising feature of the protein according tothe present invention is its ability to bind specifically to an HCVprotein, preferably an envelope protein, particularly the E2 protein.

[0011] On the basis of this specificity and other features describedbelow, we infer that the 24 kd protein of the invention is a cellularreceptor for HCV.

[0012] We have shown that the protein is ubiquitous in humans amongstthe cell types we have tested, paralleling the situation found for manyother viruses of this type (such as vaccinia virus and influenza virus).

[0013] We have shown that the protein is species specific in a mannerwhich matches the species specificity of HCV itself.

[0014] Our experiments have shown that the 24 kd protein is functionallyunglycosylated. Treatment with glycosidases does not affect the abilityof the 24 kd protein to bind to the E2 protein and does not appearsignificantly to reduce the molecular weight. We infer therefore that,if the protein is glycosylated at all, glycosylation must be restrictedto a small number of sugar moieties and is not necessary for functionalactivity of the protein.

[0015] Our experiments have also shown that the protein is atransmembrane protein, again suggesting that it is a cellular receptor.

[0016] Finally, experiments with cell lines hyperexpressing the proteinindicate that such cells are prone to aggregation suggesting that theprotein may be an adhesion molecule of some form.

[0017] The 24 kd protein may be in its naturally occurring form, albeitisolated from its native environment, or may be modified, provided thatit retains the functional characteristic of at least binding to the E2protein of HCV. For example, the 24 kd protein may be modifiedchemically to introduce one or more chemical modifications to the aminoacid structure. It may include modifications of the amino acid sequenceinvolving one or more insertions, deletions or replaced amino acids. Itmay, for example, be truncated by the removal of a functional part ofthe transmembrane domain to facilitate ready production by recombinantDNA means in a suitable mammalian host cell (6).

[0018] The protein of the present invention may be purified from cellsexhibiting binding to an HCV protein, such as the E2 protein.

[0019] According to the present invention there is provided a processfor the preparation of a protein according to the invention or afunctionally equivalent variant or fragment thereof comprising the stepof culturing cells exhibiting binding to an HCV protein and purifyingfrom a cell preparation a protein according to the invention.

[0020] The cells may be transformed or untransformed mammalian cells andare suitably human cells.

[0021] The cells may be screened for binding to an HCV protein usingfluorescence flow cytometry or any other suitable assay. For example,the present description provides the information necessary to producethe 24 kd protein or a functionally equivalent variant or fragmentthereof which then itself be used to assay for further cells carryingthe protein.

[0022] The cell preparation may be a cell membrane preparation but ispreferably a plasma cell membrane preparation.

[0023] Preferably the cells are selected and cloned to providehyperexpression of the protein of the present invention.

[0024] We have discovered that the protein is precipitated by ammoniumsulphate at between 33 and 50% of saturation.

[0025] Preferably, therefore, the cell preparation is subjected to anammonium sulphate precipitation purification step employing ammoniumsulphate at between 33 and 50%. Suitably a first precipitation isconducted at less than 33% and precipitated material discarded followedby precipitation of the desired material at between 33 and 50%, mostpreferably 50%.

[0026] Preferably, the purification involves at least one step ofhydrophobic interaction chromatography.

[0027] We have also discovered that the protein is stable to acetoneprecipitation, thereby providing a still further characterisation and auseful purification process step.

[0028] Most preferably in optimised form, the process of purificationcomprises the steps of:

[0029] i) preparing a plasma cell membrane preparation of mammaliancells selected for hyperexpression of the 24 kd protein of theinvention,

[0030] ii) subjecting the preparation to ammonium sulphate precipitationat less than 33% saturation and retaining the supernatant,

[0031] iii) subjecting the supernatant to ammonium sulphateprecipitation at between 33 and 50% saturation and retaining theprecipitate, and

[0032] iv) resuspending the precipitate and subjecting it to hydrophobicinteraction chromatography

[0033] As an alternative to purification from wild-type cell lines, theprotein of the invention or a functionally equivalent variant orfragment thereof may be made by any suitable synthetic process includingchemical synthesis. Suitably, the protein or a functionally equivalentvariant or fragment thereof is made by expression of a gene encoding theprotein in a suitable host cell or animal.

[0034] According to a further aspect of the invention, there is provideda method for treating an infection of HCV comprising administering to apatient an amount of the protein of the invention or a functionallyequivalent variant or fragment thereof effective to reduce theinfectivity of the virus.

[0035] Since the infection mechanism of HCV appears to depend, in part,upon the availability of a cell surface receptor, making available asoluble form of the protein of the invention will act as an antagonistof binding of HCV to the cellular receptor thus reducing or preventingthe infection process and thereby treating the disease.

[0036] A suitable soluble form of the protein of the invention mightcomprise, for example, a truncated form of the protein from which thetransmembrane domain has been removed either be a protein cleavage stepor, by design, in a chemical or recombinant DNA synthesis.

[0037] Alternatively, a hybrid particle comprising at least oneparticle-forming protein, such as hepatitis B surface antigen or aparticle-forming fragment thereof, in combination with the protein ofthe invention or a functionally equivalent variant or fragment thereofcould be used as an antagonist of binding of HCV to the cellularreceptor.

[0038] According to a further aspect of the invention, there is provideda pharmaceutical composition comprising a protein of the invention or afunctionally equivalent variant or fragment thereof, optionally as apharmaceutically acceptable salt, in combination with a pharmaceuticallyacceptable carrier.

[0039] The pharmaceutical composition may be in any appropriate form foradministration including oral and parenteral compositions.

[0040] A process is also provided for making the; pharmaceuticalcomposition, in which a protein of the present invention or afunctionally equivalent variant or fragment thereof is brought intoassociation with a pharmaceutically acceptable carrier.

[0041] According to a further aspect of the invention, there is provideda protein of the invention or a functionally equivalent variant orfragment thereof for use as a pharmaceutical.

[0042] According to a further aspect of the invention, there is providedthe use of a protein of the invention or a functionally equivalentvariant or fragment thereof in the manufacture of a medicament for thetreatment of an HCV infection.

[0043] The ability of a protein of the invention or a functionallyequivalent variant or fragment thereof to bind to HCV permits the use ofthe protein or a functionally equivalent variant or fragment thereof asa diagnostic for HCV infection, for example in an ELISA or RIA.

[0044] A soluble form of the protein could, for example, be used in anELISA form of assay to measure neutralising antibodies in serum.

[0045] According to a further aspect of the invention, there is providedan assay for HCV antibodies in a serum sample comprising the step ofallowing competitive binding between antibodies in the sample and aknown amount of an HCV protein for binding to a protein of the inventionor a functionally equivalent variant or fragment thereof and measuringthe amount of the known HCV protein bound.

[0046] Preferably, the protein of the invention or functionallyequivalent variant or fragment thereof is immobilised on a solid supportand the HCV protein, which may suitably be E2 HCV envelope protein,optionally recombinant E2 protein, is labelled, suitably enzymelabelled.

[0047] In an assay of this form, competitive binding between antibodiesand the HCV protein for binding to the protein of the invention resultsin the bound HCV protein being a measure of antibodies in the serumsample, most particularly, neutralising antibodies in the serum sample.

[0048] A significant advantage of the assay is that measurement is madeof neutralising antibodies directly (i.e those which interfere withbinding of HCV envelope protein to the cellular receptor). Such anassay, particularly in the form of an ELISA test has considerableapplications in the clinical environment and in routine blood screening.

[0049] Also, since the assay measures neutralising antibody titre, theassay forms a ready measure of putative vaccine efficacy, neutralisingantibody titre being correlated with host protection.

[0050] In a further aspect of the invention, there is provided adiagnostic kit comprising the protein of the invention or a functionallyequivalent variant or fragment thereof. Preferably the kit also containsat least one HCV labelled HCV protein, optionally enzyme labelled.

[0051] The protein of the invention or a functionally equivalent variantor fragment thereof may be used to screen for chemical compoundsmimicking the HCV surface structure responsible for binding to the HCVreceptor.

[0052] According to a further aspect of the invention, there is provideda method for screening chemical compounds for ability to bind to theregion of HCV responsible for binding to a host cell, comprisingmeasuring the binding of a chemical compound to be screened to a proteinof the invention or a functionally equivalent variant or fragmentthereof.

[0053] This aspect of the invention encompasses the products of thescreening process whether alone, in the form of a pharmaceuticallyacceptable salt, in combination with one or more other active compoundsand/or in combination with one or more pharmaceutically acceptablecarriers. Processes for making a pharmaceutical composition are alsoprovided in which a chemical compound identified by the process of theinvention is brought into association with a pharmaceutically acceptablecarrier.

[0054] The chemical compound may be an organic chemical and may containamino acids or amino acid analogues. Preferably however the chemicalcompound is a polypeptide or a polypeptide which has been chemicallymodified to alter its specific properties, such as the affinity ofbinding to the protein of the invention or a functionally equivalentvariant or fragment thereof or its stability in vivo.

[0055] At present, the only available animal model is the chimpanzee,which is a protected species. Experiments on such animals pose a numberof difficulties which together result in a very considerable expense (aone year experiment with one chimpanzee can cost $70,000). Compared tothis, a mouse model would be far more acceptable. Unfortunately, asdescribed below the HCV receptor, whilst ubiquitous in humans and foundin chimpanzees, is absent in other mammals. A transgenic mammal, forexample a mouse, carrying the HCV receptor on the cell surface would beof great benefit to HCV research and the development of vaccines.

[0056] According to a further aspect of the invention, there is provideda transgenic non-human mammal, suitably a mouse, carrying a transgeneencoding a protein of the invention or a functionally equivalent variantor fragment thereof.

[0057] The transgenic animal of the invention may carry one or moreother transgenes to assist in maintaining an HCV infection.

[0058] There is also provided a process for producing a transgenicanimal comprising the step of introducing a DNA encoding a protein ofthe invention or a functionally equivalent variant or fragment thereofinto the embryo of a non-human mammal, preferably a mouse.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059]FIG. 1 is a schematic diagram describing an assay in which HCVreceptor-binding ligands bind to receptors on HCV receptor target cellsand are measured by first binding rabbit anti-HCV antibody and then bybinding a labelled anti-rabbit IgG-FITC F(ab′) fragment prior to cellseparation by FACScan analysis.

[0060]FIG. 2 is a computer-generated histogram depicting the results ofa FACScan analysis of binding of HCV protein to haematopoietic cells(MOLT-4, Jurkat, K562, Daudi, EBV-B) and epithelial cells (Hela,Adenocarcinoma and Huh 7) resulting from binding with HCV proteins(filled curve unlabelled control, open curve labelled). The plot is ofcell population against fluorescence intensity.

[0061]FIG. 3 is a computer-generated histogram depicting the results ofa FACScan analysis of purified RA, purified RO, cord blood purif. RA,cord-blood RA pha stim., KC3 T-cell clone (TCC) and SAG S9 TCC, whichwere tested for binding to recombinant HCV E2 protein expressed in CHOcells (filled curve unlabelled control, open curve labelled). The plotis of cell population against fluorescence intensity.

[0062]FIG. 4 is a set of computer-generated histograms depicting theresults of a FACScan analysis of the binding of E2 CHO to MOLT-4 cellswith and without treatment with beta mercaptoethanol (BSH) an S—Slinkage reducing agent (filled curve unlabelled control, open curvelabelled). The plot is of cell population against fluorescenceintensity.

[0063]FIG. 5 is a set of computer-generated histograms depicting theresults of a FACScan analysis of the binding of E2 CHO to MOLT-4 cellswith and without treatment with Endo-H, a deglycosylating enzyme (filledcurve unlabelled control, open curve labelled). The plot is of cellpopulation against fluorescence intensity.

[0064]FIG. 6 is a western-blot of membranes prepared from MOLT-4 cellsand solubilized in different buffers (see page 22 for lanedescriptions).

[0065]FIG. 7 is a western blot of plasmatic membrane from MOLT-4 cells(see page 23 for lane descriptions).

[0066]FIG. 8 is a western blot of MOLT-4 and PBMC membrane proteins (seepage 23 for lane descriptions).

[0067]FIG. 9 is a western blot of MOLT 4 cells membrane proteins treatedwith N-Glycosidase F (see page 26 for lane descriptions).

[0068]FIG. 10 is a western Blot of MOLT-4 and COS-7 membraneselectrophoresed in reducing and non reducing conditions (see page for 27lane descriptions).

[0069]FIG. 11 is a western blot of an experiment demonstratingimmunoprecipitation of an E2-CHO/putative receptor complex (see page 29for lane descriptions).

[0070]FIG. 12 is a western blot of ammonium sulphate fractions fromMOLT-4 cells membranes (see page 31 for lanes).

[0071]FIG. 13 is a western blot of samples from a hydrophobicinteraction chromatography experiment with an acetone precipitation step(see page 32 for lanes).

DETAILED DESCRIPTION OF THE INVENTION

[0072] The arrangement of the detailed description is as follows:

[0073] 1. General Description 13

[0074] 2. Cellular Assay 13

[0075] 2.1. FACS analysis of cells binding to E2 13

[0076] 2.2. Effect of E2 modification on binding 15

[0077] 2.2.1. E2 reduction 16

[0078] 2.2.2. E2 deglycosylation 16

[0079] 2.3. Monoclonal antibody production 17

[0080] 3. Preparation of 24 kd putative receptor 17

[0081] 3.1. 24 kd protein preparation from MOLT-4 cells 17

[0082] 3.1.1. Membrane purification 17

[0083] 3.1.2. Plasma membrane purification 18

[0084] 3.2. Hyperexpressing MOLT-4 cells 20

[0085] 4. Characterisation of Receptor 20

[0086] 4.1. Western blot protocol 20

[0087] 4.1.1. Membrane proteins 21

[0088] 4.1.2. Plasma membrane proteins 22

[0089] 4.1.3. Western blot of PBMC cells 23

[0090] 4.2. Cell surface expression of receptor 23

[0091] 4.3. Effect of enzymes on 24 kd protein binding 24

[0092] 4.3.1. Flow cytometry 24

[0093] 4.3.2. Western blot on MOLT-4/N-Glycosidase F 26

[0094] 4.4. Effect of reducing condition on binding 26

[0095] 5. Optimising Purification 27

[0096] 5.1. Immunoprecipitation 27

[0097] 5.2. Ammonium Sulphate Fractionation 30

[0098] 5.3. Hydrophobic Interaction Chromatography 31

[0099] 5.4. Acetone precipitation 32

[0100] 6. Sequencing and cloning 33

[0101] 6.1. Amino acid sequence 33

[0102] 6.2. DNA sequence cloning and sequencing 33

[0103] 1. General Description

[0104] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of immunology,cytofluorimetry and molecular biology, which are within the skill of theart. Such techniques are explained fully in the literature (7).

[0105] The skilled person will understand and be familiar with thegeneral methods and techniques of assay design and practice. Theinvention is described herein in sufficient detail for the skilledperson to understand and repeat the experiments disclosed.

[0106] Standard abbreviations for virus and proteins are used in thisspecification. All publications, patents and patent applications citedherein are incorporated by reference. Envelope 1 (E1) and Envelope 2(E2) of HCV refer to the proteins, and fragments thereof, the nucleotidesequence of which are published (EP-A-0318216 and EP-A-0388232 citedabove). The nucleotides of the E1 and E2 genes and of the encodedproteins vary in different HCV isolates. Therefore, the E1 and E2 forany HCV isolates are identified because included in the amino acidsequences 192-383 and 384-750 respectively.

[0107] E1 and E2 have been produced by recombinant DNA techniques usingdifferent expression systems (Spaete et al and Chien et al cited above).

[0108] 2. Cellular Assay

[0109] 2.1. FACS Analysis of Cells Binding to E2

[0110] An experiment was performed with the aim of measuring the abilityof HCV protein to bind to various cell types which should have theputative HCV receptor.

[0111] Cells (10⁵/well) from the human T cell lymphoma, Molt-4(commercially available and obtainable from the American Type CultureCollection), were pelleted in 96 U-bottom microplates (Costar) bycentrifugation at 200×g for 5 minutes at 4° C. Twenty microliters of HCVproteins (CHO expressed recombinant E2 protein) diluted in PBS indifferent concentrations (from 10 μg/ml to 0.001 μg/ml) were mixed withthe pellet of Molt-4 cells and incubated at 4° C. for 60 minutes. Nonbound HCV proteins were removed by two centrifugations in PBS at 200×gfor 5 minutes at 4° C.

[0112] Cells were subsequently incubated for 30 minutes at 4° C. withvarious dilutions (from {fraction (1/10)} to {fraction (1/300000)}) ofsera from humans, chimps, rabbits or mice that were either infected withHCV or have been immunised with HCV recombinant proteins or thecorresponding pre-immune sera as control.

[0113] The cells were washed twice in PBS and incubated for 30 minuteswith the appropriate dilutions of fluorescein-isothiocyanate-conjugatedantisera (either to human IgG, or rabbit IgG, or mouse IgG).

[0114] Cells were subsequently washed in PBS at 4° C., resuspended in100 μl PBS and cell-bound fluorescence was analyzed with a FCScan flowcytometer (Becton & Dickinson). By using a dot plot display of forwardand side scatter, the machine is gated to include viable single cellsand to exclude cell debris and clumps of cells. A total of 5000 eventswere collected and analyses of the data was done by using the Lysis IIsoftware program from Becton & Dickinson. This program produceshistograms of each cell sample and calculates the mean channelfluorescence of the cell population, which directly relates to thesurface density of fluorescently labelled HCV proteins bound to thecells.

[0115] Mean fluorescence values (mean channel number) of cells incubatedwith or without HCV proteins and with immune or preimmune sera werecompared. The threshold for positivity is set for each experiment byflow cytometric analysis of cells without HCV proteins bound which havebeen incubated with antisera to HCV proteins and the FITC labelledsecond antibody. A representative binding experiment is shown in FIG. 1which shows the separation achieved by flow cytometric analysis.

[0116] The experiment was also conducted with a variety of cell lines(for example haematopoietic cells other than MOLT-4 such as Jurkat,K562, Daudi, EBV-B (B-cell line transformed with Epstein-Barr virus andepithelial cells such as Hela, Adenocarcinoma and Huh 7) to identifycells capable of binding HCV proteins and therefore cells that have theputative receptor(s) for HCV following the protocol described above. Itwill be appreciated that repetition of this experiment is not necessaryfor the working of the present invention, but serves to prove theubiquitous nature of the putative receptor (most of the cells are, inany event commonly available and were, in fact, obtained from the ATCC).The results were shown in FIG. 2, together with those for MOLT-4 anddemonstrate that the specific binding of E2 to cells is widespread,suggesting that the HCV receptor is ubiquitous.

[0117] In a similar series of experiments, purified RA, purified RO,cord blood purif. RA, cord blood RA pha stim., KC3 TCC and SAG S9 TCC,which were tested for binding to recombinant HCV E2 protein expressed inCHO cells (E2-CHO) and found to bind confirming that binding occurs tonon-transformed cell lines. The results are shown in FIG. 3.

[0118] 2.2. Effect of E2 Modification on Binding

[0119] The effects of modifying the recombinant HCV protein E2,expressed in CHO cells (E2-CHO) on binding to MOLT-4 cells wereinvestigated using a reducing agent and a deglycosylating enzyme.

[0120] 2.2.1. E2 Reduction

[0121] 25 μl of E2-CHO SMC-PC (130 μg/ml) in 20 mM potassium phosphate,0.1 M NaCl, pH 6.0 were buffered at pH 8.0 with 1M Tris-base (total 1μl) and then added with BSH (beta mercaptoethanol) to a finalconcentration of 500 mM; tubes were flushed with nitrogen and thereaction allowed to proceed for 100 minutes at 37° C.

[0122] Samples from above were diluted 1:20 with RPMI medium and usedfor a FACS binding assay with MOLT-4 cells as described above. The finalconcentration of BSH in the FACS assay was 12.5 mM and under theseconditions, cells were verified to be alive in previous preliminaryexperiments (over 90% of cells still alive at 50 mM BSH).

[0123] The results in FIG. 4 show that binding of MOLT-4 cells torecombinant E2 is substantially reduced on reduction withbeta-mercaptoethanol, indicating a requirement for a correct E2conformation maintained by S—S bridges.

[0124] 2.2.2. E2 Deglycosylation

[0125] 25 μl of E2-CHO SMC-PC (130 μg/ml) in 20 mM potassium phosphate,0.1 M NaCl, pH 6.0 were added with 30 μl of 0.2 M NaH₂PO₄ to a final pHof 5.5 plus SDS to a final concentration of 0.01%. Then 10 μl of Endo-Hstock was added to a final concentration of 200 mU/ml keeping the pHconstant and the resulting sample was kept at 37° C. for 20 hours. Thesample was then diluted 1:20 with RPMI and used for in a FACS bindingassay as described above

[0126] The results in FIG. 5 show that binding of MOLT-4 cells torecombinant E2 is substantially reduced on deglycosylation with Endo-H,indicating a requirement for glycosylation of E2 for binding.

[0127] 2.3. Monoclonal Antibody Production

[0128] Monoclonal antibodies were prepared using standard procedures andby immunising mice with recombinant HCV E2 produced in CHO cells(E2-CHO). Several cell lines were established which were capable ofbinding to E2-CHO as were cell lines capable of binding to E2 bound toMOLT-4 cells and cell lines capable of neutralising the binding ofE2-CHO to MOLT-4 cells.

[0129] 3. Preparation of 24 Kd Putative Receptor

[0130] 3.1. 24 Kd Protein Preparation from MOLT-4 Cells

[0131] The 24 kd protein was purified from MOLT-4 cells by membranepurification and by plasma membrane purification, the latter giving thebetter yield of protein.

[0132] 3.1.1. Membrane Purification

[0133] MOLT-4 cells were grown at 37° C., 5% CO₂ in RPMI buffered with25 mM Hepes in a growth medium containing Fetal Calf Serum (FCS—finalconcentration 5%), 1 mM glutamine, 100 μg/ml kanamicin, MEM vitamins(Gibco), 1 mM sodium pyruvate, MEM non essential amino acids (Gibco),5×10⁻⁵M β-mercaptoethanol.

[0134] Cells were harvested after reaching a density of750,000-1,000,000 cells per ml.

[0135] The growth medium containing cells was centrifuged at 300 g for10 minutes to pellet down the cells. Pelleted cells were washed threetimes in PBS Buffer (resuspended and recentrifuged).

[0136] The cell pellet was resuspended in hypotonic solution at 1 ml ofhypotonic solution per 100×10⁶ cells.

[0137] The hypotonic solution contained Tris (10 mM), NaCl (10 mM),CaCl₂ (0.2 mM), MgCl₂ (1.5 mM), PMSF (1.0 mM), aprotinin (2.0 μg/ml),pepstatin (0.7 μg/ml) and leupeptin (0.5 μg/ml).

[0138] Cells were left at 4° C. under gentle shaking for 20 minutes andthen disrupted with 25 strokes of a Potter manual homogenizer.

[0139] Membranes were recovered after sequential centrifugation of thesupernatant at 100 g for 7 minutes, 3500 g for 10 minutes and 40,000 gfor 60 minutes. The pellet obtained from the above was dissolved insuitable buffer.

[0140] The buffer used for dissolution of the 40,000 g membrane pelletcontained 1% Triton X-100 in PBS buffer pH 7.4, 8 mM Chaps in PBS bufferpH 7.4 and 4 M Urea in sodium phosphate buffer pH 7.4.

[0141] All the buffers used for membrane solubilisation containedprotease inhibitors at the concentrations reported above for thehypotonic solution and were used at a ratio of 200 μl of buffer per5×10⁸ cells.

[0142] Solubilised material was centrifuged at 1:00,000 g for 60 minutesand the supernatant kept for further use after estimation of proteincontent by BCA method.

[0143] The material obtained was subjected to analyses as describedbelow.

[0144] 3.1.2. Plasma Membrane Purification

[0145] The procedure for plasma membrane purification was based onMorre' D. J. et al. (8).

[0146] MOLT-4 cells were grown at 37° C., 5% CO₂ in RPMI buffered with25 mM Hepes in a growth medium containing Fetal Calf Serum (FCS—finalconcentration 5%), 1 mM glutamine, 100 μl/ml kanamicin, MEM vitamins(Gibco), 1 mM sodium pyruvate, MEM non essential amino acids (Gibco),5×10⁻⁵ M β-mercaptoethanol.

[0147] Cells were pelleted from culture medium and washed three timeswith PBS.

[0148] The pelleted cells were resuspended in 0.2 mM EDTA, 1 mM NaHCO₃containing the following protease inhibitors: PMSF (1.0 mM), aprotinin(2.0 μg/ml), pepstatin (0.7 μg/ml), leupeptin (0.5 μg/ml) at a ratiobetween buffer and cells of 2 ml per each 10⁸ cells.

[0149] Resuspended cells were disrupted with a Polytron homogenizerusing an S25 N10 G probe for 40 seconds at 9500 rpm. Cell disruption wasverified by optical microscope. The homogenate was centrifuged at 300 gand the resulting supernatant further centrifuged at 23,500 g for 60minutes. The resulting pellet was resuspended in 0.2 M potassiumphosphate pH 7.2 containing protease inhibitors in the ratios describedabove. The buffer volume was 1 ml each 5×10⁸ cells.

[0150] The membrane suspension was partitioned across the following twophase system: 20% (w/w) T500 Dextran 13.2 g 40% (w/w) PEG 3350 6.6 g 0.2KP, pH 7.2 0.8 ml membrane susp. 5.0 g Distilled water up to 35 g

[0151] The sample as chilled at 4° C. and the tubes were inverted 30 to40 times keeping the temperature constant. The sample was thencentrifuged on a swinging bucket rotor at 150-200 g for 5 minutes at 4°C. The upper phase was removed and five-fold diluted with 1 mM sodiumbicarbonate containing protease inhibitors. The membranes were collectedby centrifugation at 30,000 g for 30 minutes.

[0152] The pellet was dissolved in a suitable buffer and centrifuged at100,000 g for 60 minutes to eliminate undissolved material.

[0153] The material obtained was subjected to analyses as describedbelow.

[0154] 3.2. Hyperexpressing MOLT-4 Cells

[0155] A further cell line capable of hyperexpression of thecharacteristic binding ability for E2 was prepared by selecting andrecloning MOLT-4 cells binding E2 strongly. The resulting cell-lineshowed a markedly greater binding affinity for E2 than the wild-typestrain.

[0156] 4. Characterisation of Receptor

[0157] 4.1. Western Blot Protocol

[0158] The following experiments demonstrate binding of E2 to purified24 kd protein in a western blot of proteins from MOLT-4 cells purifiedfrom membranes and from plasma membranes and from peripheral bloodmononuclear cells (PBMC).

[0159] Unless otherwise indicated, all SDS-PAGE experiments wereperformed according to Laemmli et al (9), samples of solubilisedmembranes were run under non-reducing conditions and without boilingbefore each electrophoretic run.

[0160] After electrophoretic transfer (Western blot) in buffercontaining 25 mM Tris, 192 mM glycine, 20% methanol at constant electricfield of 10 Volts/cm, blotted transfer supports were saturated for 2hours in PBS buffer pH 7.4 containing 0.05% Tween 20 and 10% powderedskimmed milk at room temperature. After 1×15-minutes and 2×5 minutes,washes in PBS, 0.05% Tween 20 containing it powdered skimmed milk,transfer supports were incubated overnight with E2-CHO recombinantprotein at a concentration of 1-2 μg/ml dissolved in PBS buffercontaining 0.05% Tween 20, 1% milk, 0.02% sodium azide. Negative controltransfer supports (blotted with the same samples) were incubated for thesame time in the same buffer without E2-CHO protein.

[0161] To detect E2-CHO recombinant protein bound to the transfersupports, these were incubated with the culture supernatant of anhybridoma named 291A2 (a monoclonal antibody that recognises epitopesexposed on E2 when bound to its putative receptor) at 1:500 dilution inPBS, Tween 0.05%, milk 1% for 2 hours.

[0162] After this step, transfer supports were washed 1×15 minutes and2×5 minutes with PBS Tween 0.05% milk 1% solution. Transfer supportswere then incubated for 1 hour with biotin conjugated goat anti-mouseimmunoglobulin specific polyclonal antibody of commercial source(PharMingen, San Diego, Calif., USA) at 1:2000 dilution in thePBS/Tween/Milk solution mentioned above. After this step, transfersupports were washed 1×15 minutes and 2×5 minutes with PBS/Tween/Milk.Finally transfer supports were incubated for 1 hour withExtravidin®-Peroxidase (Sigma Immunochemicals Co., St Louis, Mo., USA)at 1:2500 dilution in PBS/Tween/Milk. Transfer supports were then washed1×15 minutes and 4×5 minutes with PBS buffer pH 7.4 containing 0.05%Tween 20. Chemiluminescent staining was performed using ECL™ westernblotting detection reagents (Amersham, UK).

[0163] 4.1.1. Membrane Proteins

[0164] A membrane preparation was prepared as described above.

[0165] Membrane pellets resulting from 40,000 g centrifugation weredissolved in buffers reported below and centrifuged at 100,000 g toremove undissolved material. Pellets were reextracted with 1% TritonX-100 in PBS pH 7.4.

[0166] Following SDS-PAGE (15 μg/lane) and blotting, the transfersupports were incubated with E2-CHO recombinant protein as describedabove.

[0167] The results are shown in FIG. 6. Lane Description 1A 4M Urea in50 mM sodium phosphate pH 7.2 2A Pellet from lane 1A sample solublizedin 1% Triton X-100 in PBS pH 7.4 3A 1% Triton X-100 in PBS pH 7.4 4APellet from lane 3A sample solubilized in 1% Triton X-100 in PBS pH 7.45A 0.01% Triton X-100 in PBS pH 7.4 6A Pellet from lane 5A samplesolubilized in 1% Triton X-100 in PBS pH 7.4

[0168] 1B to 6B are negative controls for the corresponding samples inlanes 1A to 6A.

[0169] The protein band at 24 kd is clearly visible.

[0170] 4.1.2. Plasma Membrane Proteins

[0171] A plasma membrane preparation was prepared as described above.

[0172] Plasma membranes were solubilized in PBS pH 7.4 containing 1%Triton X-100 and subjected to Laemmli SDS-PAGE. The transfer support wasincubated with E2-CHO recombinant protein.

[0173] The results are shown in FIG. 7. Lane Description 1A plasmamembranes, 10 μg total protein content 2A plasma membranes, 5 μg totalprotein content

[0174] Lanes 1B and 2B are negative controls for the correspondingsamples in lanes 1A and 2A.

[0175] The protein band at 24 kd is clearly visible.

[0176] 4.1.3. Western Blot of PBMC Cells

[0177] To assess whether the 24 kd protein could be identified in normalcells a sample of peripheral blood mononuclear cells was purified usingthe procedure described above and subjected to western blotting asdescribed above.

[0178] The results are described in FIG. 8. Lane Description ½ Molt-4membrane proteins 3 PBMC membrane proteins (22 μg/ml) 4 PBMC membraneproteins (44 μg/ml)

[0179] The negative control lanes are marked “−E2 CHO”

[0180] 4.2. Cell Surface Expression of Receptor

[0181] Employing the protocols described above, various cell types wereanalysed using FACscan and western blotting for the presence of the 24kd protein putative HCV receptor.

[0182] The results are depicted below: FACS W B HCV T and B lympho human+++ +++ +++ Monocytes human +++ +++ HeLa human ++ +++ Gastric carcinomahuman ++ +++ Hepatoma cells human +++ +++ +++ Myoblastoma human + −Fresh liver cells Green − − − monkeys Lymphomonocytes rabbit − − Freshliver cells rabbit − − Any cells mouse − −

[0183] These results demonstrate that the species distribution of the 24kd protein matches that of HCV infection susceptibility.

[0184] 4.3. Effect of Enzymes on 24 Kd Protein Binding

[0185] 4.3.1. Flow Cytometry

[0186] The biochemical nature of the cell surface component (receptor)that mediates attachment of E2 CHO envelope protein to Molt 4 cells wasinvestigated. Pretreatment of Molt 4 cells with V. choleraeneuraminidase, which has a α-2,3 specificity does not reduce E2 CHObinding.

[0187] The proteinaceous nature of the receptor was demonstrated whencells pretreated with proteases abolished binding capability of E2 CHOwhereas phospholipase treatment of cells did not affect the binding,suggesting that the cellular attachment proteins were notglycosylphosphatilylinositol anchor linked. The E2 binding site on Molt4 cells was sensitive to all protease used, which included both serineproteases, such as trypsin, and a thiol protease, such as papain.

[0188] The results of proteolytic treatment demonstrated the involvementof membrane proteins in envelope protein binding and were as follows:Fluorescence intensity Treatment Concentration (% of control) Control100 Pronase E  10 μg/ml 36 Pronase E 100 μg/ml 34 Trypsin 100 μg/ml 33Papain 100 μg/ml 42 Phospholipase C  3 U/ml 100 (from Bacillus cereus)Phospholipase C  25 U/ml 96 Neuraminidase  50 mU/ml 100

[0189] Cells (10⁶ ml⁻¹) were incubated for 60 min at 37° C. in RPMI1640/Hepes medium plus the enzymes indicated above. The cells werecentrifuged, resuspended in fresh medium and incubated with E2 CHOprotein (3 μg/ml). Purified anti E2 CHO monoclonal antibody (1.5 μg/ml)was used as a second step. Purified phycoerythrin-labelled rabbit antimouse antibody (5 μg/ml) was used as a third step reagent. A total of5000 cells per sample was evaluated with a FACScan flow cytometer.

[0190] Enzymes were used at a concentration that did not affect cellviability as measured by propidium iodine exclusion during FACSanalysis.

[0191] Fluorescence was recorded as arbitrary units (channel numbers) ona logarithmic scale and median intensities determined. Data fromindividual experiments were normalized with respect to the fluorescenceof unstained control samples- and value are expressed as percentages ofthe fluorescence intensities in the stained control samples.

[0192] 4.3.2. Western Blot on MOLT-4/N-Glycosidase F

[0193] Peptide N-Glycosidase F treatment was performed incubatingmembrane proteins (50 μg) overnight at 37° C. in phosphate buffer pH 7,4, 25 mM EDTA plus enzyme (50 U/ml) and successively loaded on 12% SDSPAGE.

[0194] To show the activity N-Glycosidase F enzyme (PNGase F), ascontrol, gp 120 polypeptide was used in the same experiment (data notshown).

[0195] The results are shown in FIG. 9. Lane Description 1 +/ve control2 treated and boiled membrane 3 untreated and boiled membrane 4 treatedmembrane 5 untreated membrane

[0196] These results show that treatment of a MOLT 4 membranepreparation with Peptide-N-glycosidase F, which hydrolyzes all N-linkedglycanes, does not abolish E2 CHO binding.

[0197] 4.4. Effect of Reducing Condition on Binding

[0198] A western blot of MOLT-4 and COS-7 membranes, prepared asdescribed above, were electrophoresed in reducing and non reducingconditions to establish the requirement or otherwise for disulphidebridges in the 24 kd protein, by measuring the binding of E2-CHO to thetransfer support.

[0199] Transfer supports were incubated with E2-CHO recombinant proteinas described above.

[0200] The results are shown in FIG. 10. Lane Description 1A COS-7membranes in non reducing SDS Laemmli buffer 2A MOLT-4 membranes in nonreducing SDS Laemmli buffer 3A COS-7 membranes in SDS Laemmli buffercontaining 5% β-SH 4A MOLT-4 membranes in SDS Laemmli buffer containing5% β-SH

[0201] 1B to 4B are negative controls for the corresponding lanes 1A to4A.

[0202] 5. Optimising Purification

[0203] 5.1. Immunoprecipitation

[0204] Membrane Proteins Solubilization

[0205] A membrane preparation from 400 million MOLT-4 cells (A2A6subclone) was treated with 200 μl of PBS buffer, pH 7.4, containingCHAPS 7.5 mM and the following protease inhibitors in μl/ml: PMSF 35,aprotinin 2, pepstatin 0.7, leupeptin 0.5. After treatment with theabove buffer, the resulting suspension was centrifuged at 100,000 g for1 hour and the clear supernatant underwent immunoprecipitationexperiments. The final protein concentration based on BCA protein assay(Pierce, USA) was 2.7 mg/ml.

[0206] Incubation with E2 Recombinant Envelope Protein

[0207] 200 μl of membrane protein solution (2.7 mg/ml) in PBS-CHAPS wereadded to 15 μl of CHO-produced E2 (Batch P4) solution. The stockconcentration of E2-P4 protein was 130 μl/ml and its final concentrationin the protein membrane solution was 9.75 μg/ml.

[0208] The mixture was kept overnight under stirring at 4° C.

[0209] Incubation with Rabbit Anti-E2 Antisera

[0210] The resulting solution was divided into two aliquots of 100 μland each was mixed with 5 μl of preimmune and postimmune antiserum froma rabbit (R#1) previously immunized with E2 protein. The final dilutionof antisera was 1:20. Incubation was performed for 1 hour at 4° C.

[0211] Addition of Protein-A Sepharose CL-4B

[0212] Protein-A Sepharose CL-4B resin (Pharmacia, Sweden) wasextensively washed with PBS containing 7.5 mM CHAPS at pH 7.4, and 30 μlof compact slurry (capacity of matrix is 20 mg of human Ig per ml ofslurry) were added to each 100 μl sample resulting from the step above.Incubation was performed under stirring for 1 hour at 4° C.

[0213] The samples were centrifuged to pellet down the resin and thesupernatant was removed, mixed with Laemmli-Buffer (without reducingagent) and kept for SDS-PAGE. The resin pellet was washed twice with 500μl of PBS-CHAPS (10 min each wash at 4° C.) and then the pellet wastreated with 50 μl of Laemmli Buffer containing 5M urea. The resultingsupernatant was subjected to SDS-PAGE.

[0214] SDS-PAGE and Immunoblot

[0215] The samples from the steps above, that is, supernatant containingmaterial not absorbed on Protein-A matrix (SN) and material desorbedfrom Protein-A matrix (ProtA) from both preimmune and postimmuneantisera, were loaded on SDS-PAGE gel in non reducing buffer and withoutheating. After the run, the gels were electroblotted on nitrocellulosepaper in 20% methanol Tris-Glycine buffer and were subjectedimmunostaining as described above. Incubation with E2 protein wasperformed overnight using E2 SMC-PC at 1.73 μg/ml in PBS 0.05% Tween20.1% milk.

[0216] The samples loaded on SDS-PAGE were:

[0217] A) Supernatant (material not retained by Prot-A) from Preimmuneantiserum,

[0218] B) Prot-A desorbed material from PREimmune antiserum,

[0219] C) Supernatant from POSTimmune antiserum, and

[0220] D) Prot-A desorbed material from POSTimmune antiserum

[0221] Three sets of these samples were loaded on gel, one was staineddirectly on gel the other two underwent immunostain, one incubated withE2 the other as negative control.

[0222] The nitrocellulose transferred support was incubated with E2-CHOSMC-PC recombinant protein at 1.73 g/ml followed by 291A2 hybridomaculture supernatant (containing a monoclonal antibody that recognisesepitopes exposed on E2 when bound to its putative receptor),biotinylated polyclonal anti-mouse Ig antibodies and peroxidase labelledExtravidin™ (Sigma Immunochemicals, USA). Chemiluminescent stainingobtained with ECL-Luminol (Amersham, GB), exposure 1 minute.

[0223] The results are shown in FIG. 11. Lane Description 1A molecularweight standard 2A empty 3A sample incubated with preimmune rabbitserum - supernatant 4A sample incubated with preimmune rabbit serum -Protein-A bound 5A sample incubated with postimmune rabbit serum -supernatant 6A sample incubated with postimmune rabbit serum - Protein-Abound

[0224] The negative controls employed the nitrocellulose membraneincubated with 291A2 hybridoma culture supernatant followed bybiotinylated polyclonal anti-mouse Ig antibodies and peroxidase labelledstreptavidin. 1B to 4B correspond to 3A to 6A respectively.

[0225] 5.2. Ammonium Sulphate Fractionation

[0226] Membranes were prepared as reported in the membrane preparationprotocol from MOLT-4 cells and solubilized in PBS buffer pH 7.4containing 8 mM CHAPS. The protein concentration estimated on the basisof BCA assay ranges from 1.8 and 2.5 mg/ml.

[0227] Solubilized membranes were mixed with an ammonium sulphate (AS)saturated solution in a volume sufficient to obtain 25% saturation ofammonium sulphate (i.e. the final concentration of AS is 25% of thestarting saturated solution). The sample was allowed to stand in meltingice for 2 hours and then centrifuged at 15800 g for 30 minutes.

[0228] The supernatant was mixed with AS saturated solution to a finalsaturation of 50%. The sample was allowed to stand for 2 hours onmelting ice and then filtered on a Spin-X™ centrifuge filter unit(Costar, Cambridge, Mass., USA) for 15 minutes at 4° C.

[0229] The precipitates obtained above were dissolved in suitablebuffers and undergo further treatment.

[0230] The pellets were redissolved in PBS pH 7.4 containing 10M ureaand underwent Laemmli SDS-PAGE. The volumes Of AS fractions were loadedin such a way that the amount of putative receptor should be comparablein different samples.

[0231] The transfer support was incubated with E2-CHO recombinantprotein as described above.

[0232] The results are shown in FIG. 12 and show that precipitation ofp24 occurs in the range of 33 to 50% of saturation. Lane Description 1AStarting membranes 2A 20% AS fraction 3A 33% AS fraction 4A 43% ASfraction 5A 50% AS fraction 6A 60% AS fraction

[0233] 5.3. Hydrophobic Interaction Chromatography

[0234] 1.5 ml of solubilized membranes from MOLT-4 cells (proteinconcentration 2.5 mg/ml) were pre-fractionated at 25% of saturation ofammonium sulphate and the supernatant from this step was brought to 50%saturation of AS. The precipitate obtained was resuspended in 200 μl ofPBS containing ammonium sulphate at 25% of saturation. The undissolvedmaterial was pelleted by centrifugation at 15800 g for 30 minutes. Thesupernatant obtained was incubated with 200 μl of Phenyl-Sepharosematrix (Pharmacia, Uppsala, Sweden), previously equilibrated in PBS pH7.2 containing AS at 25% of saturation, for 2 hours at room temperature.

[0235] The non retained material was recovered by filtering on a Spin-X™centrifuge filter units (Costar, USA).

[0236] The matrix of Phenyl-Sepharose was washed twice with 100 μl ofPBS, 25% AS saturation and once with 300 ml of the same buffer. Thematrix was then eluted with PBS pH 7.4 (200 μl) and then with PBS, pH7.4 containing 20 MeOH. Finally, the matrix was treated with 40 μl ofnon reducing Laemmli buffer.

[0237] Samples containing ammonium sulphate (i.e. non retained and washmaterial) were dialysed against 8M urea in PBS, pH 7.4.

[0238] All samples underwent SDS-PAGE analysis and Western Blot.

[0239] 5.4. Acetone Precipitation

[0240] 50 μl of membranes from MOLT-4 cells solubilized in 8 mM CHAPS inPBS, pH 7.4 were mixed with 200 μl of acetone.

[0241] The sample was centrifuged at 15800 g for 15 minutes and thesupernatant was discarded. The obtained precipitate was dissolved in nonreducing Laemmli sample buffer and underwent SDS-PAGE and Western Blot.The transfer supports incubated with E2-CHO recombinant protein asdescribed above.

[0242] The results of the combined HIC and acetone precipitationexperiment are shown in FIG. 13. Lane Description 1A Starting membranes(16 μl total protein content) 2A material non retained on matrix 3A wash4A material eluted with PBS, pH 7.4 5A material eluted with PBS, pH 7.4containing 20% Methanol 6A material eluted with Laemmli Buffer 7Amembranes solubilized in Triton 1% PBS, pH 7.4 (26 μg total protein) 8Aacetone precipitate from sample 7A

[0243] Samples from 1B to 8B correspond to samples from 1A to 8A

[0244] These experiments show that ammonium sulphate precipitatedmaterial can be redissolved in suitable conditions and undergohydrophobic interaction chromatography. The 24 kd HCV putative receptorprotein binds to Phenyl Sepharose and can be recovered from this matrix.

[0245] The Membrane extract can be precipitated with acetone withoutloss of binding capacity of HCV putative receptor.

[0246] 6. Sequencing and Cloning

[0247] 6.1. Amino Acid Sequence

[0248] The amino acid sequence of the −24 kd protein may be elucidatedeither by inference from the cloned DNA or by microsequencing of proteinprepared by one of the processes described above. Based upon themolecular weight of the protein (and the knowledge that, if glycosylatedit is only glycosylated to a small extent) it is expected that theprotein will have approximately 210-230 amino acids (allowing theaverage of 110 daltons per amino acid).

[0249] 6.2. DNA Sequence Cloning and Sequencing

[0250] The DNA sequence of the 24 kd protein may be determined by one ofa number of techniques known to the art, such as λgt11 “shotgun” cloningwhere a DNA library is produced, suitably from a hyperexpressingcell-line (see above) and fragments of DNA were caused to express inprokaryotic or eukaryotic cell, the products being screened usingantibodies to the 24 kd protein or by binding to recombinant E2-CHO.

[0251] Once identified, the DNA encoding the 24 kd protein may be usedto produce large quantities of the protein which, as a result of itsbinding to HCV may prove useful in an assay for HCV infection or for themanufacture of a medicament for treating HCV infection.

[0252] Alternatively, the DNA may be used to prepare transgenic animalsbearing the 24 kd protein which may then serve as animal models for HCVinfection.

[0253] It will be understood that the invention is described above byway of example and modifications within the scope and spirit of theinvention may be made without the need for undue experiment or theexercise of inventive ingenuity.

REFERENCES

[0254] 1. European patent application EP-A-0318216

[0255] 2. European patent application EP-A-0388232

[0256] 3. Choo et al PNAS USA (1991) δ 2451-2455

[0257] 4. Chien, D. Y. et al PNAS USA (1992) 89 10011-10015

[0258] 5. Spaete, R. R. et al Virology (1992) 188 819-830

[0259] 6. Gething et al Nature [needs complete reference]

[0260] 7. “Flow Cytometry” in Methods of Cell Biology, 1990 Vol. 33Academic Press San Diego

[0261] 8. Morre' D. J. et al., Methods Enzymol. (1994) 228, 448-450

[0262] 9. Laemmli et al Nature (1970) 27 680

1. A protein having a molecular weight of about 24 kD and capable ofspecifically binding to a protein of hepatitis C virus, or afunctionally equivalent variant or fragment thereof.
 2. A protein or afunctionally equivalent variant or fragment thereof according to claim 1which is functionally unglycosylated.
 3. A protein or a functionallyequivalent variant or fragment thereof according to claim 1 or 2 whereinthe protein is a transmembrane protein.
 4. A process for the preparationof a protein or a functionally equivalent variant or fragment thereofaccording to any one of claims 1 to 3 comprising the step of culturingcells exhibiting binding to an HCV protein and purifying from a cellpreparation a protein according to any one of claims 1 to
 3. 5. Aprocess according to claim 4 wherein the cell preparation is a plasmacell membrane preparation.
 6. A process according to claim 4 or 5wherein the cells are selected and cloned to provide hyperexpression ofthe protein according to any one of claims 1 to
 3. 7. A processaccording to any one of claims 4 to 6 wherein the cell preparation issubjected to an ammonium sulphate precipitation purification stepemploying ammonium sulphate at between 33 and 50%
 8. A process accordingto any one of claims 4 to 7 wherein the purification involves at leastone step of hydrophobic interaction chromatography.
 9. A processaccording to any one of claims 4 to 8 wherein the process involves atleast one step of acetone precipitation
 10. A process according to anyone of claims 4 to 8 wherein comprising the steps of: i) preparing aplasma cell membrane preparation of mammalian cells selected forhyperexpression of the 24 kd protein of the invention, ii) subjectingthe preparation to ammonium sulphate precipitation at less than 33%saturation and retaining the supernatant, iii) subjecting thesupernatant to ammonium sulphate precipitation at between 33 and 50%saturation and retaining the precipitate, and iv) resuspending theprecipitate and subjecting it to hydrophobic interaction chromatography11. A method for treating an infection of HCV comprising administeringto a patient an amount of a protein according to any one of claims 1 to3 or a functionally equivalent variant or fragment thereof effective toreduce the infectivity of the virus.
 12. A pharmaceutical compositioncomprising a protein according to any one of claims 1 to 3 or afunctionally equivalent variant or fragment thereof, optionally as apharmaceutically acceptable salt, in combination with a pharmaceuticallyacceptable carrier.
 13. A process for preparing a pharmaceuticalcomposition, in which a protein according to any one of claims 1 to 3 ora functionally equivalent variant or fragment thereof is brought intoassociation with a pharmaceutically acceptable carrier.
 14. A proteinaccording to any one of claims 1 to 3 or a functionally equivalentvariant or fragment thereof for use as a pharmaceutical.
 15. Use of aprotein according to any one of claims 1 to 3 or a functionallyequivalent variant or fragment thereof in the manufacture of amedicament for the treatment of an HCV infection.
 16. An assay for HCVantibodies in a serum sample comprising the step of allowing competitivebinding between antibodies in the sample and a known amount of an HCVprotein for binding to a protein according to any one of claims 1 to 3or a functionally equivalent variant or fragment thereof and measuringthe amount of the known HCV protein bound
 17. A diagnostic kitcomprising the protein according to any one of claims 1 to 3 or afunctionally equivalent variant or fragment thereof.
 18. A method forscreening chemical compounds for ability to bind to the region of HCVresponsible for binding to a host cell, comprising measuring the bindingof a chemical compound to be screened to a protein according to any oneof claims 1 to 3 or a functionally equivalent variant or fragmentthereof.
 19. A transgenic non-human mammal, carrying a transgeneencoding a protein according to any one of claims 1 to 3 or afunctionally equivalent variant or fragment thereof.
 20. A process forproducing a transgenic animal comprising the step of introducing a DNAencoding a protein according to any one of claims 1 to 3 or afunctionally equivalent variant or fragment thereof into the embryo of anon-human mammal, preferably a mouse.